Inhibition of 3C protease from human rhinovirus strain 1B by peptidyl bromomethylketonehydrazides.

Autor: Kati WM; Pharmaceutical Products Division, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, Illinois, 60064-3500, USA. warren.kati@abbott.com, Sham HL, McCall JO, Montgomery DA, Wang GT, Rosenbrook W, Miesbauer L, Buko A, Norbeck DW
Jazyk: angličtina
Zdroj: Archives of biochemistry and biophysics [Arch Biochem Biophys] 1999 Feb 15; Vol. 362 (2), pp. 363-75.
DOI: 10.1006/abbi.1998.1038
Abstrakt: The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in an Escherichia coli strain which also overexpressed the rare argU tRNA. The protease was isolated from inclusion bodies, refolded, and exhibited a kcat/Km = 3280 M-1 s-1 using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides. Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease with kinact/Kinact values as high as 23,400 M-1 s-1. The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to the kinact/Kinact rate constants for bromomethylketonehydrazide inhibition of 3C protease. Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145-154, a region which contains the active site cysteine-148 residue. The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture.
(Copyright 1999 Academic Press.)
Databáze: MEDLINE
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